Inflammatory bowel disease (IBD) is characterized by poorly healing, refractory mucosal ulceration. Investigation from our group has identified profound alterations in microvascular function in both forms of IBD (Crohn's disease (CD), ulcerative colitis (DC)), where arterioles isolated from chronically inflamed intestine demonstrate an acquired loss of vasodilation capacity. This microvascular dysfunction will lead to impaired tissue perfusion, which may contribute to the chronic, poorly healing mucosal damage which characterizes both CD and UC. The mechanisms underlying microvascular dysfunction in IBD involve an alteration in oxyradical balance, where the microvascular endothelium fails to generate nitric oxide (.NO), and instead produces sustained levels of superoxide anion. We seek to specifically define the enzymatic and molecular mechanisms underlying this acquired disturbance in microvascular endothelial function using both freshly isolated human gut microvessels, as well as primary cultures of control and disease specific intestinal microvascular endothelial and smooth muscle cells. Thus our central hypothesis is: Excess and sustained superoxide generation and impaired .NO production underlies altered endothelial function in chronic IBD inflammation, leading to diminished vasodilatory capacity and impaired intestinal healing. This hypothesis will be tested by the following two specific aims: Specific Aim A. Determine superoxide and ROS production in normal control, involved and uninvolved IBD intestinal microvessels and the functional effect on microvascular dilation. 1) Determine superoxide and ROS production in control, involved and uninvolved IBD microvessels. 2) Determine the enzymatic mechanisms responsible for superoxide and ROS production and their role in microvascular dilation in normal control, involved and uninvolved IBD microvessels. 3) Characterize microvascular expression of the enzymes/isozymes involved in superoxide and ROS generation and degradation from normal control, involved and uninvolved IBD tissues. Specific Aim B. Determine the mechanisms of superoxide and ROS generation in human intestinal microvascular endothelial cells (HIMEC) and vascular smooth muscle cells (VSMC) isolated from normal control, involved and uninvolved IBD patient tissues. 1) Determine superoxide and ROS generation in control and IBD HIMEC. 2) Determine the enzymatic source of superoxide and ROS generation from control and IBD HIMEC. 3) Determine eNOS expression, activation status and function in control and IBD HIMEC. 4) Determine MnSOD expression in control and IBD HIMEC. 5) Determine superoxide and ROS generation and enzymatic sources in VSMC isolated from control and IBD intestinal arterioles. The experiments in this application are designed to determine the alterations in oxyradical/.NO balance as well as enzymatic mechanisms underlying altered ROS generation, which underlie microvascular dysfunction in chronic intestinal inflammation. Findings from this proposal will form the foundation for a mechanistic understanding of microvascular dysfunction in human chronic inflammation, and may directly lead to novel therapeutic approaches to improve microvessel function, tissue perfusion and mucosal healing in IBD patients.